The invention relates to neurotrophic factor polypeptides, nucleic acids encoding neurotrophic factor polypeptides, and antibodies that bind specifically to neurotrophic factors.
Neurotrophic factors are naturally-occurring proteins which promote survival, maintain phenotypic differentiation, prevent degeneration, and enhance the activity of neuronal cells and tissues. Neurotrophic factors are isolated from neural tissue and from non-neural tissue that is innervated by the nervous system, and have been classified into functionally and structurally related groups, also referred to as families, superfamilies, or subfamilies. Among the neurotrophic factor superfamilies are the fibroblast growth factor, neurotrophin, and transforming growth factor-xcex2 (TGF-xcex2) superfamilies. Individual species of neurotrophic factors are distinguished by their physical structure, their interaction with their composite receptors, and their affects on various types of nerve cells. Classified within the TGF-xcex2 superfamily (Massague, et al,. 1994, Trends in Cell Biology, 4:172-178) are the glial cell line-derived neurotrophic factor ligands (xe2x80x9cGDNFxe2x80x9d; WO 93/06116, incorporated herein by reference), which include GDNF, persephin (xe2x80x9cPSPxe2x80x9d; Milbrandt et al., 1998, Neuron 20:245-253, incorporated herein by reference) and neurturin (xe2x80x9cNTNxe2x80x9d; WO 97/08196, incorporated herein by reference). The ligands of the GDNF subfamily have in common their ability to induce signalling through the RET receptor tyrosine kinase. These three ligands of the GDNF subfamily differ in their relative affinities for a family of neurotrophic receptors, the GFRxcex1 receptors.
Due to the affects of neurotrophic factors on neuronal tissue, there remains a need to identify and characterise additional neurotrophic factors for diagnosing and treating disorders of the nervous system.
This invention relates to a novel neurotrophic factor herein called xe2x80x9cneublastin,xe2x80x9d or xe2x80x9cNBN.xe2x80x9d Neublastin is classified within the GDNF subfamily because it shares regions of homology with other GDNF ligands (see Tables 3 and 4, infra) and because of its ability to interact with RET (see, e.g., Airaksinen et al., Mol. Cell. Neuroscience, 13, pp. 313-325 (1999)), neublastin is a novel and unique neurotrophic factor. Unlike other GDNF ligands, neublastin exhibits high affinity for the GFRxcex13-RET receptor complex and unique subregions in its amino acid sequence.
A xe2x80x9cneublastin polypeptide,xe2x80x9d as used herein, is a polypeptide which possesses neurotrophic activity (e.g., as described in Examples 6, 7, 8, and 9) and includes those polypeptides which have an amino acid sequence that has at least 70% homology to the human xe2x80x9cneublastinxe2x80x9d polypeptides set forth in AAxe2x88x9295-AA105 of SEQ. ID. NO. 2, AA1-AA105 of SEQ. ID. NO. 2, AAxe2x88x9297-AA140 of SEQ. ID. NO. 4, AAxe2x88x9241-AA140 of SEQ. ID. NO. 4 (xe2x80x9cproxe2x80x9d), AA1-AA140 of SEQ. ID. NO. 4, AAxe2x88x9280-AA140 of SEQ. ID. NO. 9 (xe2x80x9cwild typexe2x80x9d prepro), AAxe2x88x9241-AA140 of SEQ. ID. NO. 9 (pro), AA1-AA140 of SEQ. ID. NO. 5 (mature 140AA), AA1-AA116 of SEQ. ID. NO. 6 (mature 116AA), AA1-AA113 of SEQ. ID. NO. 7 (mature 113AA), AA1-AA140 of SEQ. ID. NO. 10 (mature 140AA), AA1-AA116 of SEQ. ID. NO. 11 (mature 116AA), AA1-AA113 of SEQ. ID. NO. 12 (mature 113AA), and variants and derivatives thereof. In addition, this invention contemplates those polypeptides which have an amino acid sequence that has at least 70% homology to the murine xe2x80x9cneublastinxe2x80x9d polypeptides set forth in AA1-AA224 of SEQ. ID. NO. 16.
Preferably, the C-terminal sequence of the above identified neublastin polypeptides has an amino acid sequence as set forth in AA72-AA105 of SEQ. ID. NO. 2 (i.e., AA107-AA140 of SEQ. ID. NO. 9), more preferably AA41-AA105 of SEQ. ID. NO. 2 (i.e., AA76-AA140 of SEQ. ID. NO. 9), or the amino acid sequence set forth in AA191-AA224 of SEQ. ID. NO. 16.
Also, it is preferable that the neublastin polypeptide retain the 7 conserved Cys residues that are characteristic of the GDNF family and of the TGF-beta super family.
Preferably, the neublastin polypeptide has an amino acid sequence greater than 85% homology, most preferably greater than 95% homology, to the foregoing sequences (i.e., AAxe2x88x9295-AA105 of SEQ. ID. NO. 2, AA1-AA105 of SEQ. ID. NO. 2, AAxe2x88x9297-AA140 of SEQ. ID. NO. 4, AA1-AA140 of SEQ. ID. NO. 4, AAxe2x88x9241-AA140 of SEQ. ID. NO. 4, AAxe2x88x9280-AA140 of SEQ. ID. NO. 9 (xe2x80x9cwild typexe2x80x9d prepro), AAxe2x88x9241-AA140 of SEQ. ID. NO. 9 (pro), AA1-AA140 of SEQ. ID. NO. 5 (mature 140AA), AA1-AA116 of SEQ. ID. NO. 6 (mature 116AA), AA1-AA113 of SEQ. ID. NO. 7 (mature 113AA), AA1-AA140 of SEQ. ID. NO. 10 (mature 140AA), AA1-AA116 of SEQ. ID. NO. 11 (mature 116AA), AA1-AA113 of SEQ. ID. NO. 12 (mature 113AA)), and AA1-AA224 of SEQ. ID. NO. 16.
A xe2x80x9cneublastin nucleic acid,xe2x80x9d as used herein, is a polynucleotide which codes for a neublastin polypeptide. Accordingly, an isolated neublastin nucleic acid is a polynucleotide molecule having an open reading frame of nucleotide codons that, were it to be exposed to the appropriate components required for translation, would encode, or code for, a neublastin polypeptide. Neublastin nucleic acids of the invention may be RNA or DNA, e.g., genomic DNA, or DNA which is complementary to and/or transcribed from, a neublastin mRNA (xe2x80x9ccDNAxe2x80x9d). Thus, a neublastin nucleic acid of the invention further includes polynucleotide molecules which hybridize with specificity, under high stringency hybridization conditions, to a polynucleotide that codes for a neublastin polypeptide. This invention also relates to nucleic acid primers that are useful in identifying, isolating and amplifying polynucleotides that encode neublastin polypeptides, or fragments thereof. In certain embodiments of the invention, certain of these primers are neublastin-specific probes useful for hybridization to a neublastin nucleic acid, but not to nucleic acids coding for the other members of the GDNF family. By xe2x80x9cspecificxe2x80x9d, xe2x80x9cspecificityxe2x80x9d, or xe2x80x9cspecificallyxe2x80x9d, is meant an ability to hybridize with neublastin nucleic acid and inability to hybridize with non-neublastin nucleic acids, including an inability to hybridize to nucleic acids that code uniquely for the GDNF ligands (e.g., GDNF, persephin, and neurturin).
In another embodiment, a neublastin nucleic acid of the invention is one that is identified as being complementary to a polynucleotide that codes for a neublastin polypeptide, either by having a complementary nucleic acid sequence or demonstrating that it hybridizes with specificity at high stringency hybridization conditions to a polynucleotide that codes for neublastin. Particular neublastin nucleic acids include, without limitation, the nucleic acid sequences shown herein and designated SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 29 and SEQ ID NO: 30 as well as primers SEQ ID NOS: 17-28, 31 and 32. A neublastin nucleic acid of the invention further includes a unique subregion, or fragment, of a neublastin nucleic acid, including without limitation the nucleic acid fragments shown in FIG. 8.
The neublastin nucleic acids of the invention may be used to express a neublastin polypeptide, e.g., by expressing a neublastin polypeptide in vitro, or by administering a neublastin nucleic acid to an animal for in vivo expression. Neublastin nucleic acids may be included within a nucleic acid vector, e.g., an expression vector or a cloning vector. A neublastin nucleic acid may, but need not of necessity, be maintained, reproduced, transferred, or expressed as part of a nucleic acid vector. A recombinant expression vector containing a neublastin polynucleotide sequence can be introduced into and/or maintained within a cell. Cells hosting a neublastin vector may be prokaryotic. Alternatively, a neublastin nucleic acid can be introduced into a eukaryotic cell, e.g., a eukaryotic cell that contains the appropriate apparati for post-translational processing of a polypeptide into a mature protein, and/or the appropriate apparati for secreting a polypeptide into the extracellular environment of the cell.
The invention further features a neublastin neurotrophic factor, xe2x80x9cneublastin.xe2x80x9d Neublastin may be in the form of a polypeptide, or may be a multimer of two or more neublastin polypeptides, e.g., a neublastin dimer. Neublastin polypeptides are associated as multimers by intermolecular structural associations known to those skilled in the art, including without limitation cysteine-cysteine interaction, sulfhydryl bonds, and noncovalent interactions. Particular neublastin polypeptides include, without limitation, an amino acid sequence disclosed herein and designated SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 16.
A neublastin polypeptide of the invention is useful for treating a defect in a neuron, including without limitation lesioned neurons and traumatized neurons. Peripheral nerves that experience trauma include, but are not limited to, nerves of the medulla or of the spinal cord. Neublastin polypeptides are useful in the treatment of neurodegenerative disease, e.g., cerebral ischemic neuronal damage; neuropathy, e.g., peripheral neuropathy, Alzheimer""s disease, Huntington""s disease, Parkinson""s disease, amyotrophic lateral sclerosis (ALS). Neublastin polypeptides are further contemplated for use in the treatment of impaired memory, e.g., memory impairment associated with dementia.